Optimal amounts of ascorbic acid (vitamin C) for maintaining human health a unknown. As a unique means to address this problem we developed the concep of in situ, in which the principles of reaction kinetics are applied to vitamin C dependent reactions in situ. We studied catecholamine biosynthes in secretory vesicles to test the principles of in situ kinetics. We found that ascorbic acid provides single electrons via transmembrane electron transfer to the intra- vesicular enzyme dopamine beta-monooxygenase. Ascorbic acid outside vesicles transfers electrons to reduce the free radic intermediate semidehydroascorbic acid within vesicles; the free radical is formed as ascorbic acid within vesicles transfers single electrons to the monooxygenase. Electron transfer in situ occurs at V(max) under physiologi conditions. These experiments validated the principles of in situ kinetics We previously discovered that ascorbic acid is accumulated by human neutrophils in mM concentration. We recently found that the physiologic structural analog glucose exquisitely regulates ascorbic acid transport. Experiments are underway to characterize the mechanism of glucose regulatio We discovered that human lymphocytes also contain mM concentrations of ascorbic acid. To learn vitamin C function, experiments using differential hybridization and subtraction cloning are underway. Ascorbic acid accumulation in human fibroblasts was characterized as a prelude to understanding how different concentrations of the vitamin regulate proline hydroxylation. Clinical goals are to learn how much vitamin C is found in humans as a function of ingestion, so that ascorbic acid dependent reaction can occur. In addressing prerequisite issues we found that ascorbic acid i normal human plasma and serum is free and not protein bound. Ascorbic acid circulates only in the reduced form. With these results an intensive clinical trial was proposed and approved to learn how ascorbic acid ingesti regulated plasma and tissue concentrations.